Christiane Moisand

ORL1, a novel member of the opioid receptor family : cloning, functional expression and localization

Selective PCR amplification of human and mouse genomic DNAs with oligonucleotides encoding highly conserved regions of the δ‐opioid and somatostatin receptors generated a human DNA probe (hOP01, 761 bp) and its murine counterpart (mOP86, 447 bp). hOP01 was used to screen a cDNA library from human brainstem. A clone (named hORL1) was isolated, sequenced and found to encode a protein of 370 amino acids whose primary structure displays the seven putative membrane‐spanning domains of a G protein‐coupled membrane receptor. The hORL1 receptor is most closely related to opioid receptors not only on structural (sequence) but also on functional grounds: hORLl is 49–50% identical to the murine μ‐, δ‐ and κ‐opioid receptors and, in CHO‐K1 cells stably transfected with a pRc/CMV:hORLl construct, ORL1 mediates inhibition of adenylyl cyclase by etorphine, a ‘universal’ (nonselective) opiate agonist. Yet, hORLl appears not to be a typical opioid receptor. Neither is it a somatostatin or σ (N‐allylnormetazocine) receptor. mRNAs hybridizing with synthetic oligonucleotides complementary to mOP86 are present in many regions of the mouse brain and spinal cord, particularly in limbic (amygdala, hippocampus, septum, habenula,⋯) and hypothalamic structures. We conclude that the hORL1 receptor is a new member of the opioid receptor family with a potential role in modulating a number of brain functions, including instinctive behaviours and emotions.

Comparison of the structure‐activity relationships of nociceptin and dynorphin A using chimeric peptides

The aim of the present study was to delineate the functional domains of nociceptin (noc), a neuropeptide which is structurally related to dynorphin A (dyn). The binding and biological potencies towards the nociceptin (ORL1) and dynorphin A (κ‐opioid) receptors of twenty dyn/noc and noc/dyn hybrid peptides were compared with those of the parent heptadecapeptides. Replacement of as many as eleven residues in the C‐terminus of dynorphin by the corresponding nociceptin sequence has no significant effect on binding and biological activity towards the κ‐opioid receptor. In marked contrast, replacement of as few as six residues (RKLANQ) in the C‐terminus of nociceptin by the corresponding dynorphin sequence (LKWDNQ) dramatically impairs both affinity and activity towards the ORL1 receptor. This clearly indicates that the two neuropeptides have different functional architectures, despite the dual structural homology of both ligands and receptors. Moreover, the recombinant peptide approach led us to identify hybrids whose sequences differ only at positions 5 and 6 and displaying opposite or no receptor selectivity. One contains the dynorphin Leu5‐Arg6 sequence and prefers the κ‐opioid receptor, whereas the other comprises the nociceptin Thr5‐Gly6 sequence and prefers the ORL1 receptor. A third, containing the mixed dynorphin/nociceptin Leu5‐Gly6 sequence, does not discriminate between the two types of receptor.

Replacement of Gln280 by His in TM6 of the human ORL1 receptor increases affinity but reduces intrinsic activity of opioids

The ORL1 ( pioid eceptor‐ ike) receptor is the G protein‐coupled receptor whose amino acid sequence is closest to those of opioid receptors. Residues that are conserved in ORL1 and the three types of opioid receptor, but also a residue, His in the sixth putative transmembrane (TM6) helix, which is present in all opioid receptor types but absent in ORL1, appear to play a key role in receptor recognition and/or activation. Here we have sought to create an opioid binding pocket in the non‐opioid ORL1 receptor by replacing residue Gln280 in its TM6 by the corresponding His residue of opioid receptors. The mutation affects neither the affinity of nociceptin ‐ the natural ORL1 agonist ‐ for the receptor, nor the potency of nociceptin to inhibit adenylyl cyclase via ORL1. In contrast, we find that a few opioid ligands, the agonists lofentanil, etorphine and dynorphin A, and especially the antagonists diprenorphine and nor‐BNI, bind the mutant Q280H receptor with substantially (5‐ to > 100‐fold) higher apparent affinity than they do the wild‐type receptor. Moreover, lofentanil and etorphine no longer act as pure agonists, as they do at the native ORL1 receptor, but are endowed with clear antagonist properties at the mutant receptor. The mutation Q280H, which increases affinity while decreasing intrinsic activity of opioids at ORL1, emphasizes the importance of the His residue for opioid recognition and activation.

Apparent precoupling of κ- but not μ-opioid receptors with a G protein in the absence of agonist

Abstract Rabbit and guinea-pig cerebellum membranes contain a very high (> 80%) proportion of μ- and κ-opioid receptors, respectively. Rabbit (μ) and guinea-pig (κ) cerebellum membranes wre (i) labeled either with the opiate agonist. [ 3 H]etorphine (K d = 0.1–0.2 nM), or with the opiate antagonist. [ 3 diprenorphine (K d = 0.1 nM), in the absence or presence of Na + and/or 5′-guanylylimidodiphosphate (GppNHp), (ii) solubilized with digitonin (1%, w : v) and (iii) the radioactivity in the soluble extracts analyzed by ultracentrifugation in sucrose gradients. In the soluble extracts from rabbit cerebellum (μ) membranes, bound [ 3 H]etorphine sedimented faster (s 20 ≅ 12S) that bound [ 3 H]diprenorphine (10s), while is those from guinea-pig cerebellum (κ) membranes, bound [ 3 H]etorphine and bound [ 3 H]diprenorphine sedimented at the same position (12S). Na + selectively decreased recovery of the bound tritiated agonist in the two soluble preparations. When they had been generated in the presence of GppNHp but in the absence of Na + , the [ 3 etorphine complexes of the μ- and κ-opioid receptors as well as the [ 3 Hdiprenorphine complex of the κ-opioid receptor were all recovered at position 10S, indicating that GppNHp had induced a decrease of the apparent molecular size of the two types of opioid receptors. these data are interpreted in terms of μ- and κ-opioid receptors being capable of physically interacting with a G protein (GTP binding regulatory protein) yet, unlike the μ-opioid receptor which does so only in the presence of an agonist, the κ-opioid receptor appears to be precoupled with a G protein.

Binding of Leu5-enkephalin and Met5-enkephalin to a particulate fraction from rat cerebrum.

Leucine-enkephalin and methionine-enkephalin are two pentapeptides with morphine like properties, naturally present in mammalian brain. They were first characterized by Hughes et al. [ 1 ] as H-TyrGlyGlyPheLeu-OH and H-TyrGlyGlyPheMet-OH respectively and found to behave as potent, although short lived opiates in a variety of pharmacological tests in vivo [2]. They do interact with the so-called opiate receptor (OpR) in vitro as evidenced by their ability to inhibit the binding to OpR of a variety of non-peptide opiate agonists and antagonists [3,4]. We have made use of radioactive enkephalins to demonstrate directly their interaction with the opiate receptor in vitro. It is shown in this report that both enkephalins bind with nearly equal affinities to one class of non-interacting sites in a particulate fraction from rat cerebrum. Binding of both enkephalins cannot be distinguished on the basis of its inhibition by non-peptide opiates such as levorphanol or naloxone, but is differentially affected by a variety of cations. These results are compared with those that have recently appeared in the litterature and which favor a multiplicity of enkephalin binding sites in similar preparations [2,.5,6].